Niclosamide is a Negative Allosteric Modulator of Group I Metabotropic Glutamate Receptors: Implications for Neuropathic Pain

Article abstract of DOI:10.1007/s11095-016-2027-9

Purpose: Novel therapeutics are greatly needed that target specific pathological receptors and pathways involved in Neuropathic Pain (NP). Extending our previous work published in this Journal on Group I metabotropic glutamate receptor (mGluR) modulators, we now investigate the therapeutic potential of niclosamide in modulating aberrant glutamate transmission in NP. Method: Calcium mobilization assays and cross-receptor selectivity experiments are conducted to characterize the pharmacological activity of niclosamide. A focused series of niclosamide analogues is then prepared to elucidate key structural determinants that emerged from computational molecular modeling analysis on drug-receptor interactions. Finally, niclosamide and a carbamate derivative are studied to assess their efficacy in an NP-evoked mechanical hyperalgesia model in rats. Results: Niclosamide is a low-nanomolar allosteric antagonist of Group I mGluRs with high selectivity for Group I over homologous Group III mGluRs. The phenolic hydroxyl group of niclosamide forms a crucial hydrogen bond with mGluR1/5. Its bioactive coplanar conformation is further stabilized by the nitro substituent on the B ring and an intramolecular bond. Mechanical hyperalgesia in NP rats is reversed by niclosamide through three different dosing routes. Conclusion: To our knowledge, this is the first report of the salicylanilide class of compounds as potential treatments for NP.

Full text of DOI:10.1007/s11095-016-2027-9

Pharm Res
DOI 10.1007/s11095-016-2027-9
RESEARCH PAPER
Niclosamide is a Negative Allosteric Modulator of Group I
Metabotropic Glutamate Receptors: Implications
for Neuropathic Pain
Ni Ai1 & Richard D. Wood2 & Eric Yang2 & William J. Welsh3
Received: 9 June 2016 /Accepted: 22 August 2016
#
Springer Science+Business Media New York 2016
ABSTRACT
intramolecular bond. Mechanical hyperalgesia in NP rats is
reversed by niclosamide through three different dosing routes.
Conclusion To our knowledge, this is the first report of the
salicylanilide class of compounds as potential treatments for
NP.
Purpose Novel therapeutics are greatly needed that target
specific pathological receptors and pathways involved in
Neuropathic Pain (NP). Extending our previous work pub-
lished in this Journal on Group I metabotropic glutamate
receptor (mGluR) modulators, we now investigate the thera-
peutic potential of niclosamide in modulating aberrant gluta-
mate transmission in NP.
.
KEY WORDS group I metabotropic glutamate receptors
.
neuropathic pain niclosamide
Method Calcium mobilization assays and cross-receptor se-
lectivity experiments are conducted to characterize the phar-
macological activity of niclosamide. A focused series of
niclosamide analogues is then prepared to elucidate key struc-
tural determinants that emerged from computational molec-
ular modeling analysis on drug-receptor interactions. Finally,
niclosamide and a carbamate derivative are studied to assess
their efficacy in an NP-evoked mechanical hyperalgesia model
in rats.
ABBREVIATIONS
CNS
IASP
Central nervous system
International Association for the Study of Pain
mGluR Metabotropic Glutamate receptor
MPEP 2-methyl-6-(phenylethynyl)pyridine
NAM
NP
Negative allosteric modulator
Neuropathic pain
Results Niclosamide is a low-nanomolar allosteric antagonist
of Group I mGluRs with high selectivity for Group I over
homologous Group III mGluRs. The phenolic hydroxyl
group of niclosamide forms a crucial hydrogen bond with
mGluR1/5. Its bioactive coplanar conformation is further
stabilized by the nitro substituent on the B ring and an
PAM
PSN
SAM
Positive allosteric modulator
Partial sciatic nerve
Silent allosteric modulator
INTRODUCTION
According to the International Association for the Study of
Pain (IASP) Taxonomy working group, neuropathic pain
(NP) is defined as Bpain caused by a lesion or disease of the
somatosensory nervous system^ (1). About 6–8% of the gen-
eral population is estimated to be affected by NP (2). NP
syndromes can significantly influence the quality of life and
debilitate the physical functions of patients. However, treat-
ments for NP are few in number, of limited effectiveness, and
beset by side effects. To enhance understanding of the disease
and discover new therapeutic options, several pathophysiolog-
ical mechanisms have been identified to play an important
role in NP, such as dysregulated glutamate signaling in the
central nervous system (CNS) (3).
Electronic supplementary material The online version of this article
(doi:10.1007/s11095-016-2027-9) contains supplementary material, which is
available to authorized users.
* William J. Welsh
welshwj@rwjms.rutgers.edu
1
Pharmaceutical Informatics Institute, College of Pharmaceutical Sciences,
Zhejiang University, 866 Yuhangtang Road, Hangzhou, Zhejiang 310058,
People’s Republic of China
2
Snowdon, Inc., Princeton, New Jersey 08540, USA
3
Department of Pharmacology, Rutgers-Robert Wood Johnson Medical
School, Rutgers University, 661 Hoes Lane West, Staged Research
Building (room SRB-124), Piscataway, New Jersey 08854, USA

Ai, Wood, Yang and Welsh
The excitatory amino acid glutamate is ubiquitous in cen-
tral and peripheral nervous mechanisms. Malfunction of glu-
tamate messaging has been implicated in a variety of CNS
and peripheral disorders. Of particular importance is the role
of glutamate in pain (4,5). It is known that attenuation of
glutamate release, or antagonism of certain glutamate recep-
tors, can modulate pain (6). The metabotropic glutamate re-
ceptors (mGluRs), which bind to glutamate then modulate
synaptic transmission, are divided into three sub-groups:
Group I, II, and III. These mGluRs are members of the class
C G-protein coupled receptors (GPCRs) with structures char-
acterized by a seven transmembrane (TM) domain and an
extracellular domain responsible for orthosteric ligand bind-
ing. Classical pharmacological modulators, which largely tar-
get the orthosteric binding site of GPCRs where the endoge-
nous ligand (e.g., glutamate) performs its regulatory role, rep-
resent about 40% of currently marketed drugs across all major
therapeutic areas. Despite the tremendous record of success in
GPCR drug discovery, evolutionary conservation of the
orthosteric binding site among GPCRs poses a great challenge
for drug selectivity. In addition, chronic activation of certain
GPCRs by agonists is not tolerated (7). Since the proposal of
allosterism fifty years ago (8), allosteric modulators have
attracted considerable interest due to better receptor subtype
selectivity compared with compounds binding to the
orthosteric site (7,9–11). Moreover, the activity of allosteric
modulators is dependent on the presence of endogenous ago-
nist, which can reduce the risk of receptor oversensitization.
With respect to their modes of action, allosteric ligands can be
separated into three categories: negative allosteric modulators
(NAMs) that inhibit the receptor; positive allosteric modula-
tors (PAMs) that activate the receptor; and silent allosteric
modulators (SAMs) that exert no effects but physically occupy
the allosteric binding site. A plethora of basic and translational
discovery research has focused on identification of allosteric
modulators of GPCRs, which has led to two approved drugs
in the market functioning through this unique mechanism
while several more drug candidates are now in the clinical
development (11).
diseases (12,13). Recent results have revealed that Group I
mGluR NAMs are effective at relieving NP. The mGluR1
allosteric antagonists developed at Pfizer and the mGluR5
allosteric antagonists SIB-175715 and 2-methyl-
6-(phenylethynyl)pyridine (MPEP) (14,15) have demonstrated
substantial efficacy in rodent models of NP. These compounds
have found utility as research probes, but as yet have fallen
short as therapeutic agents. Using an in silico-in vitro-in vivo drug
repositioning procedure developed by us previously (16),
niclosamide and nitazoxanide emerged as two approved drugs
with potential Group I mGluR antagonistic activity from an
approved drug library. Niclosamide is an off-patent
antihelmintic drug with an excellent safety profile, which is
indicated for the treatment of intestinal worms by uncoupling
mitochondria of the parasitic worm. This drug recently has
attracted considerable interest as a treatment for viral infec-
tions (17,18), cancers (19–21), Type 2 diabetes (22), and other
serious pathologies. However, until now there has been no
report of the modulating activity of niclosamide on GPCR
functions. Inspired by the excellent safety profile of
niclosamide and its modulating effects on Group I mGluRs,
we have now characterized the activity profile of niclosamide
against six mGluR subtypes and investigated niclosamide in a
preclinical model as an oral treatment for NP. In an effort to
improve the bioavailability of niclosamide via prodrug design,
we also considered a derivative of niclosamide in the animal
study. Both niclosamide and its derivative relieved
hyperalgesia induced by mechanical injury in NP rats, which
attests to its utility as a starting point for the development of
NP therapeutics. Additionally, the structure of niclosamide
can be a useful pharmacological tool for elucidating the role
of mGluR on CNS functions.
METHODS
Chemistry
Commercially available salicylic acids and anilines were
coupled in hot xylenes in the presence of PCl3 to furnish
niclosamide analogues (Scheme 1) (23). R1–R4 substituents
are defined in Figs. 1, 2, 3 and 4. Reduction of niclosamide
with Zn dust in methanol and acetic acid followed by salt
formation gave amino salicylanilide hydrochloride 6. The car-
bamate derivative of niclosamide was obtained through
Ample studies have confirmed that dysregulated signaling
of the Group I mGluRs, which comprise mGluR1 and
mGluR5, is observed in many pathological neural processes
including NP. Extensive efforts by industry and academic
groups have been spent on identification of allosteric modula-
tors of these two receptors as therapeutics for many related
Scheme 1 Preparation of 1–5
and 7

Niclosamide for Neuropathic Pain
Fig. 1 (a) Chemical structure of
niclosamide; (b) Chemical
structures of representative
modulators with cross-receptor
activities between mGluR1 and
mGluR4.
a
OH
b
N
O
O
O
N
H
N
N
N
H
O
Cl
O
Cl
(-)-PHCCC (mGluR1NAM;mGluR4 PAM) VU0405623 (mGluR1PAM;mGluR4PAM)
treatment of niclosamide with Dimethylcarbamyl chloride to
provide acylated derivative 9. Compound 8 was prepared
through the reaction of 3-chlorobenzoyl chloride with 2-
chloro-4-nitroaniline in a mixture of THF and CH2Cl2 in
the presence of N,N-diisopropylethylamine (DIEA) and 4-
dimethylaminopyridine (DMAP). Detailed information on
chemical synthesis was provided in Supplemental
Information.
compounds were obtained at ≥95% purity (HPLC-MS).
Niclosamide was purchased from Sigma-Aldrich (St. Louis,
MO, USA).
BIOLOGY
In Vitro mGluR1 and mGluR5 Functional Assay
Preparation of Compounds
Synthesis of Potential Group I mGluR Modulators
For the in vitro assays, niclosamide and its analogues were
dissolved in dimethyl sulfoxide (DMSO) to a stock solution
of 10 mM with dilutions in assay buffer. Positive controls
(glutamic acid, JNJ16259685, and MPEP) were provided by
the testing company.
Unless otherwise stated, all solvents and reagents were used as
received from vendors. H NMR spectra were measured at
1
either 400 MHz (Varian) or 500 MHz (Varian Inova AS500)
in DMSO-d6 or CDCl3. HPLC-MS analyses were carried out
with a Shimadzu LC-MS 2020 using a Phenomenex CHO-
8463 C18 column (50 × 3.0 mm) with a gradient of 10%
acetonitrile: 90% water (0.1% formic acid) to 100% acetoni-
trile (0.1% formic acid) over 5 min. Retention times (TR) are
reported in minutes (min). Mass spectra (ESI) are reported in
positive (m/z+) and/or negative (m/z-) mode. The calculated
exact mass is denoted as EM. Unless otherwise stated, all
EXPERIMENTS
An aequorin cell line expressing the human recombinant
mGluR1 or mGluR5 receptor was utilized to test in vitro
mGluR1
mGluR5
Fig. 2 Dose response curve of niclosamide for group I mGluRs.

Ai, Wood, Yang and Welsh
Fig. 3 Detailed interactions of niclosamide with mGluR1 (a) and mGluR5 (b). The Group I NAM in the crystal structures is colored in olive, and the carbon
atoms in niclosamide are colored in yellow.
activities of virtual hit compounds. This functional assay was
performed by Euroscreen Inc. (Belgium) and a brief proce-
dure is described as follows. The expression of human mGluR
in this cell line was controlled by an inducible promoter
through doxycycline. Prior to the test, the mGluR1 or
mGluR5 cells were grown in an antibiotic-free media supple-
mented with doxycycline (600 ng/mL) for 18 h, then detached
by gentle flushing with PBS-EDTA (5 mM EDTA). The cells
were recovered by centrifugation and re-suspended in assay
buffer (HBSS, 2.1 mM CaCl2, 3 μg/mL glutamate-pyruvate
transaminase, 4 mM MEM sodium pyruvate, 0.1% BSA
protease-free). Cells were incubated with the light-emitting
coelenterazine-h at room temperature for at least 4 h. Test
compound (60 μL) was added into the cell suspension and
incubated together for at least 3 min, 30 μL of glutamate
solution was then added with a concentration that gives an
80% of maximal response by glutamate saturation (EC80).
The resulting emission of light was recorded using a
Hamamatsu Functional Drug Screening System 6000
(FDSS 6000). The wells containing 100 μM digitonin or a
saturating concentration (20 μM) of adenosine triphosphate
were used to standardize the emission of the recorded light
Fig. 4 Structure-activity
relationships of niclosamide
analogues.

Niclosamide for Neuropathic Pain
(determination of the B100% signal^) across plates and across
different experiments. Percentages of inhibition were calculat-
ed on the basis of the activation induced by the agonist gluta-
mate at its EC80. Each experiment was carried out three times
in three replicates. Dose–response data were analyzed with
SigmaPlot software using nonlinear regression applied to a
sigmoidal dose–response model.
EXPERIMENTS
These experiments were performed by Eurofins Inc. (Dayton
NJ, USA). The partial sciatic nerve ligation (25) (PSNL) model
of neuropathic pain was used to produce neuropathic
hyperalgesia in rats. Partial ligation of the left sciatic nerve is
performed on animals under enflurane/O.sub.2 inhalation
anesthesia. Following induction of anesthesia, the left thigh
of the rat is shaved and the sciatic nerve exposed at high thigh
level through a small incision and is carefully cleared of sur-
rounding connective tissues at a site near the trocanther just
distal to the point at which the posterior biceps semitendinosus
nerve branches off of the conunon sciatic nerve. A 7-0 silk
suture is inserted into the nerve with a 3/8 curved, reversed-
cutting mini-needle and tightly ligated so that the dorsal 1/3
to 1/2 of the nerve thickness is held within the ligature. The
wound is closed with a single muscle suture (7-0 silk) and a
Michelle clip. Following surgery, the wound area is dusted
with antibiotic powder. Sham-treated rats undergo an identi-
cal surgical procedure except that the sciatic nerve is not ma-
nipulated. Following surgery, animals are weighed and placed
on a warm pad until they recover from anesthesia. Animals
are then returned to their home cages until behavioral testing
begins. The animal is assessed for response to noxious me-
chanical stimuli by determining paw withdrawal threshold
(PWT) for the left rear paw of the animal immediately prior
to and 1, 3, 6 and 24 h after drug administration. PWT
expressed in grams, which is used as an index of mechanical
hyperalgesia, was assessed by pressure stimulation method as
described by Randall and Selitto. Increasing pressure exerted
by Randall Selitto paw pressure device (UGO Basile, SRL
Biological Research Apparatus, Italy) is applied to the left hind
paw of the rat and withdrawal of left paw or vocalization
response was taken as indicator for the nociceptive threshold
(see Supplemental Information for timeline of procedure). To
determine oral ED50 value of niclosamide in PSNL model, the
drug was administered at increasing doses ranging from 18.8
to 300 mg/kg.
In Vivo NP Animal Model
Animals
All experiments were conducted in accordance with the Guide
for the Care and Use of Laboratory Animals (National
Research Council, 2011) and all protocols were approved by
the Product Safety Labs Institutional Animal Care and Use
Committee. Male Sprague–Dawley rats (125–150 g weight)
were received from Ace Animals (Ace Animals, Inc.,
Boyertown, PA). The animal room was temperature con-
trolled and had a 12-h light/dark cycle. The animals were
fed with Purina Rodent Chow #5012 and acclimated to the
facility for 6 days prior to the study. Prior to the experiments,
the animals were randomly separated into four groups (n = 6)
(sham group, vehicle control group, positive control group
and dosed group).
Preparation of Compounds and Administration Procedures
Niclosamide was dissolved in the vehicle solution (9%
DMSO/27% Cremophor/64% saline) for intrathecal (IT)
administration (24) in a 10 μL injection volume. Then the
rat was lightly anesthetized and placed to flex the lower lum-
bar vertebrae. After palpating the vertebral processes, a 26
gauge needle was inserted through a vertebral interspace.
The presence of a tail flick was considered validation of the
needle placement; the solution was then injected.
Niclosamide and 9 were dissolved in the vehicle solution
(20% DMSO/80% Cremophor) for intraperitoneal (IP) injec-
tion in a 5 mL/kg injection volume. The high dose of
niclosamide was determined by dose of the drug used in IT
route and the low dose was about one-fourth high dose.
Niclosamide and 9 were formulated with 0.5% carboxy-
methylcellulose for oral dosing in a 10 mL/kg volume. Oral
dosage of niclosamide was calculated as three-fold dose used
in IP injection, which is approximately one-sixth the reported
oral LD50 (2500 mg/kg) of niclosamide in rat and the same
concentration was applied to 9 (the carbamate derivative of
niclosamide).
DATA ANALYSIS
All data are presented as mean S.E.M. PWT was normal-
ized as percent of pre-injury baseline value for each animal at
different post-injury and post-drug treatment time points then
plotted as a function of time. Data analysis was performed
using two-way mixed model ANOVA (factors: time/drug
treatment, injury group) with Bonferroni post hoc comparison
by SigmaPlot 12.3. The AUCs of the antinociceptive effects
measured during a 6 h period were calculated by SigmaPlot
12.3 and analyzed using one-way ANOVA with Tukey’s hon-
est significant difference (HSD) post hoc test, which is also
Positive control gabapentin was prepared and administrat-
ed in the same manner as niclosamide in the experiments
using different routes of administration. All of the compounds
where tested were under blind experimental conditions.

Ai, Wood, Yang and Welsh
used for estimation the oral ED50 of niclosamide by nonlinear
regression as described previously (26).
mGluRs in addition to Group I mGluRs. To assess whether
niclosamide might exert its biological effects through other
related mGluRs, we performed an activity profiling study
using a cell line expressed with specific human recombinant
mGluRs, including mGluR1, mGluR5 or four Group III
mGluRs, viz., mGluR4, mGluR6, mGluR7, and mGluR8.
For the in vitro experiments, three different modes of action
were tested for niclosamide at a fixed concentration: agonist
(assay buffer only), PAM (cells stimulated by an EC20 concen-
tration of reference agonist) and NAM (cells stimulated by an
EC80 concentration of agonist) mode. Dose dependent re-
sponse of niclosamide against the specific mGluR subtype
then would be evaluated if this drug showed activity at a spe-
cific mode. At the test concentration (10 μM), niclosamide was
capable of antagonizing about 75% of glutamate calcium mo-
bilization response at a concentration of its EC80 in cells ex-
pressing mGluR1 or mGluR5 and ineffective under the other
two modes, suggesting it is a NAM for Group I mGluRs. The
half maximal inhibitory concentration (IC50) was subsequently
determined for this drug to be 38 nM and 62 nM for mGluR1
and mGluR5, respectively (Fig. 2). The selective mGluR1
NAM JNJ16259685 (IC50 = 6.21 nM) and the selective
mGluR5 NAM MPEP (IC50 = 28.4 nM) served as positive
controls (data not shown). These results compare well with the
corresponding reported values of 1.21 nM for JNJ16259685
and 36 nM for MPEP. Our data indicated that negligible
in vitro activity (<50% activation or inhibition) was induced
on the Group III mGluRs by this drug at 10 μM under all
three testing modes (Table I). We conclude that niclosamide
does not act through Group III mGluRs at a concentration up
to 10 μM. Taken together, these findings suggest that
niclosamide is a selective negative allosteric modulator for
Group I over Group III mGluRs.
MOLECULAR DOCKING STUDY
Recently the X-ray crystal structures of the seven transmem-
brane domain of mGluR1 (27) and mGluR5 (28) were report-
ed, both of which contain the allosteric binding site. Structures
of mGluR1 (PDB ID: 4OR2) and mGluR5 (PDB ID: 4OO9)
were downloaded from the Protein Data Bank (http://www.
pdb.org/pdb/). The Molecular Operating Environment
(MOE) software (Version 2013, Chemical Computing
Group, Montreal, Canada) was utilized to prepare and locally
energy minimize their X-ray crystal structures for docking
studies. The DOCK module of MOE was applied to dock
niclosamide into the respective allosteric binding site of each
mGluR using default docking parameters in the program.
RESULTS
In Vitro Assays to Evaluate mGluR Subtype Selectivity
of Niclosamide
Earlier studies by us have established that both nitazoxanide
and its active metabolite (tizoxanide) function as NAMs for
Group I mGluRs and show appreciable antinociceptive effects
in a NP rodent model (16). The same study also identified the
antihelmintic drug niclosamide as a Group I mGluR antago-
nist at a concentration of 10 μM in vitro. Here we have char-
acterized the pharmacological activity of niclosamide. Due to
sequence and geometry similarity shared by the three sub-
groups of mGluRs, several lines of evidence indicate that
many allosteric compounds acting at Group I mGluRs display
differential modulating effects on other mGluR subtypes, par-
ticularly mGluR4 of the Group III mGluRs (29). Figure 1
shows two representatives of these mGluR4 modulators that
share a similar scaffold with niclosamide. Furthermore, Group
III mGluR PAMs have been demonstrated to show beneficial
effects in related animal models, including NP (30). Therefore,
it is possible that niclosamide may function through Group III
Computational Modeling to Reveal Structural
Determinants of Niclosamide with Group I mGluRs
The in vitro cross subtype selectivity assay demonstrated that
niclosamide is a dual mGluR1/5 NAM. However, it is not
clear how this antihelmintic drug interacts with these two
GPCRs to modulate downstream signaling events. Recently
published X-ray crystal structures of the transmembrane
Tab le I Group III mGluR
Modulating Activities of Niclosamide
Testing mode
% of activation or inhibition against Group III mGluRs
at 10 μM
mGluR4
mGluR6
mGluR7
mGluR8
Agonist
PAM
−3.99 1.71
−13.46 1.09
21.51 0.74
−0.06 2.14
−15.34 5.60
19.21 6.06
32.46 5.83
−2.08 0.83
−15.96 4.34
16.77 3.41
38.77 4.35
NAM
−26.40 8.65
Data are presented as percentage of activity (activation or inhibition) against each mGluR subtype as mean SD for two
independent experiments done in triplicate (see Supplementary Information for additional details on biological assays)

Niclosamide for Neuropathic Pain
domain of both mGluR1 and mGluR5 now make it possible
to investigate structural determinants of ligand-receptor inter-
actions of niclosamide at the atomic level of detail. On the
basis of structural superimposition of the mGluR1 and
mGluR5 crystal structures, the allosteric site is identified for
each receptor based on the corresponding co-crystallized
NAM, which is formed primarily by transmembrane helices
5, 6, and 7. However, these two sites occupy distinct sites in the
TM domain, where the mGluR1 site is more closely posi-
tioned to the extracellular domain and the mGluR5 site is
more deeply buried inside the TM domain. An overall flat,
linear shape is observed for the allosteric binding sites of both
Group I mGluRs, which agrees well with the common planar
geometry shared by the scaffolds of most Group I NAMs (11).
Similarly, the salicylanilide core of niclosamide provides the
structural basis for its potent activity against both receptors.
Computational docking experiments were then performed on
this drug against both mGluR1 and mGluR5. Docking results
in mGluR1 revealed that the hydroxyl group in A ring of
niclosamide forms a strong (<3.5 Å) hydrogen bond with
Thr815, which is the same residue that hydrogen bonds with
the co-crystallized mGluR1 NAM FITM (Fig. 3a). The im-
portance of this Threonine residue for ligand binding is fur-
ther confirmed by other reported mGluR1 NAMs with differ-
ent scaffolds and by mutagenesis data on the receptor (12).
Additionally, extensive hydrophobic contacts with the residues
in the mGluR1 TM domain were identified to contribute to
the here nanomolar affinity of niclosamide, including Val664,
Leu757, Ile764, Phe801, and Val819. The B-ring of the drug
was shown to point toward the bottom of the pocket; however,
due to the difference in length between niclosamide and
FITM, the allosteric binding site of mGluR1 is not fully
occupied by niclosamide. Docking of niclosamide in the
allosteric binding site of mGluR5 showed near perfect
alignment between niclosamide and the co-crystallized li-
gand mavoglurant (Fig. 3b). In particular, the amide
group in niclosamide mimicked the alkyne group in
mavoglurant as the linker group, which satisfied the nar-
row channel shape of mGluR5 allosteric site at this region.
Similarly, the interactions of niclosamide with mGluR5
were stabilized by a strong hydrogen bond with Ser805
(O-H···O distance = 3.26 Å) that is known to play a key
role in small molecule binding with mGluR5. Interestingly,
an intramolecular N−H···O hydrogen bond is found in
docked poses of niclosamide in both receptors, which is
consistent with a recent report on crystal structure of this
drug (31). Taken together, the phenolic hydroxyl group of
niclosamide represents an essential structural determinant
for interaction with both mGluR1 and mGluR5. The am-
ide linker also serves an important structural role in main-
taining the molecular conformation and overall structural
rigidity of the drug to satisfy the shape of Group I
mGluRs allosteric binding sites.
Medicinal Chemistry Study to Investigate
Ligand-Receptor Interactions of Niclosamide
To better understand ligand pharmacology of niclosamide on
group I mGluRs, a focused medicinal chemistry effort was
carried out to investigate the effects induced by the alteration
of substitutions on the rings, especially the phenolic oxygen of
the core salicylanilide structure of niclosamide. Structural and
activity information of niclosamide analogues were listed in
Fig. 4. Removal of the phenolic OH group from the A ring
of niclosamide completely abolished activity of compound 1
for both mGluR1 and mGluR5. This finding is consistent with
our observations from computational modeling that this OH
group played an essential role in determining the binding of
niclosamide to both receptors. However, other substituents on
the scaffold are also important for binding affinity as suggested
by inactive compound 2 in which all other moieties were
deleted except the –OH group in A ring. Replacement of
the 5-chloro of niclosamide with methyl (3) or H (4) at A ring
moderately decreased activity at both mGluR1 and mGluR5.
The effects of modifications of the A ring were more signifi-
cant on the activities for mGluR1 than for mGluR5, as mea-
sured by the greater fold decrease of potency (increase in
IC50). The nitro group on the B-ring of niclosamide exerts
strong electron withdrawing effects, which contribute signifi-
cantly to electron delocalization of the conjugated system
through the amide linker group. In the docked pose of
niclosamide, this electronic substituent is oriented deeply to-
ward the hydrophobic TM in mGluR1 and mGluR5. While
replacement of the nitro group at the 4′ position by the less
electron withdrawing chloro group (5) decreased potency for
mGluR1 and mGluR5 slightly, replacement by an electron
donating amino group (6) eliminated mGluR1/5 activity al-
together. Likewise, replacement of Cl at the 2′-position with H
(7) led to a 10-fold and 3-fold loss of activity for mGluR1 and
mGluR5, respectively. Alteration of the electron-withdrawing
character of the B ring substituents of niclosamide led to a
profound effect on its antagonist activity. To assess the signif-
icance of the orientation of the amide linker, we synthesized
compound 8 in which the amide linker (-C(=O)-NH-O) be-
tween the two rings of niclosamide is reversed (-NH-C(=O)-).
This compound failed to show antagonist activity in the in vitro
assays of mGluR1 and mGluR5, which demonstrates that the
direction of the amide linker plays a crucial role in receptor
recognition of niclosamide.
NP Preclinical Model to Demonstrate In Vivo Efficacy
of Niclosamide and its Carbamate Derivative
Many years of use of niclosamide attest to its minimal water
solubility. This attribute raises little concern for its primary
therapeutic indication as an antihelmintic agent in the gut.
However, solubility issues are relevant to indications that

Ai, Wood, Yang and Welsh
require access to the central and peripheral nervous systems.
As a proof-of-concept study, niclosamide was administered to
partial sciatic nerve (PSN) ligated rats, a reliable preclinical
model of NP, to observe its potential to reverse mechanical
hyperalgesia through IT route that bypasses the blood–brain
barrier (BBB). Gabapentin, an approved standard of care for
NP treatment (32) which is known to bind calcium channels
and affect release of glutamate in the brain, was selected as the
positive control for the current in vivo study. Consistent with its
inhibitory effects on activation of both mGluR receptors
in vitro, administration of niclosamide (0.15 μmol, IT) mark-
edly increased the paw withdrawal threshold (PWT) in terms
of % of pre-Injury by around 120% after 1 h drug treatment.
Niclosamide showed comparable efficacy with gabapentin
which was employed as the positive control (% of pre-Injury
= 110%, 0.58 μmol, 1 h) (Fig. 5). This effect is still significant
at 3 h post-dose but had generally dissipated by 6 h post-dose.
These in vivo data demonstrated that IT delivery of
niclosamide could potently reduce mechanical hyperalgesia
caused by nerve injury, which is consistent with the roles of
mGluR1 and mGluR5 in NP.
to develop orally active therapeutics for NP, the in vivo activi-
ties of niclosamide and 9 were then evaluated through oral
administration (PO) in the same NP rodent model. Oral ga-
vage of niclosamide reduced sensitivity of mechanical pain in
test animals (Fig. 7a). The antihyperalgesic effects in rats treat-
ed with niclosamide at 300 mg/kg reached statistical signifi-
cance at 1 h post-dose. The maximal effect was reached at 1 h
post-dose with gradually diminished effects at 3 h and 6 h
post-dose. Overall, the results suggest that niclosamide
showed substantial in vivo efficacy at 300 mg/kg PO. For
niclosamide derivative 9, significant antihyperalgesia effects
at 300 mg/kg dose were lacking when compared with the
vehicle-treated group as indicated by AUC(0-6h) (Fig. 7b).
We also demonstrated that niclosamide dose-dependently
and fully relieved PSNL-induced mechanical hyperalgesia fol-
lowing PO administration, with an estimated ED50 value of
54 mg/kg (Fig. 7c).
DISCUSSION
These encouraging results for niclosamide via IT injection
in the NP rat model prompted further in vivo evaluations of the
antihyperalgesic effects of this drug and its derivative (9)
through IP route of administration. Compound 9, a carba-
mate derivative of niclosamide, lacked mGluR1 and mGluR5
inhibitory activity in vitro; however, we reasoned that it would
be converted to the biologically active parent niclosamide
in vivo. The in vivo efficacy of the derivative was compared with
niclosamide and positive control gabapentin using the PWT
model of NP in rat. The activities of these two compounds,
administered at low and high doses, to reverse neuropathic
mechanical hyperalgesia were evaluated at four different time
points (1 h, 3 h, 6 h, 24 h). All of the tested compounds pro-
duced a statistically significant reversal of hyperalgesia, at least
at one time point (Fig. 6a and b). Dosed at 75 mg/kg,
niclosamide produced an antihyperalgesic effect indicated by
the PWT to 140% of pre-Injury observed at 1 h after treat-
ment. This effect was still significant at 6 h and abated at 24 h.
Niclosamide derivative (9) was effective in the NP animal
model with the maximal PWT near 110% of pre-Injury at
3 h. The time delay of the observed analgesic effect for this
compound is likely related to cleavage of the pro-moiety to
yield bioactive niclosamide in vivo. The integrated activities
shown by AUC(0-6h) confirmed that two drugs exhibited po-
tent in vivo efficacies at their tested concentrations (Fig. 6c). In
summary, the present data indicated that pain in the NP an-
imal is ameliorated by niclosamide and its derivative via IP
administration.
Niclosamide is an FDA approved antihelmintic drug to treat
tapeworm infection in humans and other animals by
uncoupling oxidative phosphorylation in the parasites.
Recently, this drug demonstrated beneficial effects against
obesity-related type 2 diabetes through the same mechanism
in the mitochondria of the mouse liver (22). In addition, other
inhibitory mechanisms of action, such as the STAT3 signaling
pathway (33) and the Wnt/β-catenin pathway (34), are also
reported for niclosamide to account for its pharmacological
activities in cancer cell lines and xenograft models. However,
there are no reports of the effect of niclosamide on GPCRs,
the group of drug targets mediating a number of important
signal transduction and cellular processes. Glutamate plays a
key role in pathophysiological conditions in the CNS such as
pain and its receptors on synapses. The mGluRs are a group
of GPCRs that are widely distributed along the pain neuraxis
and modulate cellular mechanisms of nociceptive sensitiza-
tion. As a consequence, they are considered as attractive drug
targets to depress pain transmission. Noncompetitive Group I
mGluR antagonists have been shown with great therapeutic
potential in several NP animal models. Our preliminary
screening effort indicates that niclosamide shows promising
inhibitory activities on Group I mGluRs in cell-based assays.
Here we further characterized niclosamide in vitro and in vivo.
A major finding from the present study is that niclosamide is a
negative allosteric modulator of Group I mGluRs and displays
selectivity to Group I over the analogous Group III mGluRs.
It is noteworthy, due to the limitation of experimental testing
conditions considered herein, the activity of niclosamide on
mGluR7 (~30% activation in Table I) requires further atten-
tion. Although it is inactive according to our current threshold,
secondary assays would be required to confirm the activation of
IP injection is an accurate, reliable, and convenient dosing
route to test the biological effects of the drugs; however, it is
used exclusively on small laboratory animals and is not
intended to replicate drug administration in humans. In order

Niclosamide for Neuropathic Pain
Fig. 5 (a) In vivo efficacy data for
niclosamide against neuropathic
mechanical hyperalgesia through IT
route. (b) The integrated effects of
tested drugs that were calculated as
AUC for 0 to 6 h post-treatment. *
P < 0.05 compared with vehicle,
data are presented as mean SEM
for six animals.
mGluR7 by niclosamide, which would provide in-depth under-
standing of anti-NP effects of this drug. A limited medicinal
chemistry study was carried out to probe structural determi-
nants of niclosamide to group I mGluRs and confirm specific
ligand-protein interactions from computational docking of the
drug to crystal structures of two receptors. Three essential
chemical features are identified for NAM activity of
niclosamide, viz., the hydroxy group in the A ring, the strong
electron-withdrawing nitro group in the B ring, and the amide
linker. Additionally, the nearly flat geometry of this drug im-
posed by the extended conjugation between the two aromatic
rings and amide linker is complementary to the shape of allo-
steric binding site of Group I mGluRs. Tizoxanide (the bioac-
tive metabolite of prodrug nitazoxanide) which emerged from
our previous screening effort (16), also satisfies the same struc-
tural characteristics and exhibits weak to moderate micromolar
activity on mGluR1/mGluR5. Moreover, CPPHA, a PAM for
Group I mGluRs and Group III mGluR4, also belongs to the
salicylanilides with a rather bulky substituent at 3′ position of
the B ring. These findings suggest that the salicylanilide core is a
favorable scaffold recognized by allosteric binding sites of
mGluRs. Further SAR studies are required with a larger and
more structurally diverse array of salicylanilide analogues and
derivatives to more fully discern SAR patterns that will guide
structural optimization.
Currently available therapeutic drugs for NP are associated
with limited efficacy and a high incidence of side effects (35);
therefore, there is an urgent need to develop an alternative
medicine which can attenuate neuropathic pain effectively.
Results from in vitro studies demonstrated that niclosamide
allosterically inhibited calcium mobilization induced by en-
dogenous ligand in mGluR1/5. However, the poor solubility
of niclosamide may limit its therapeutic potential in the clinic.
A carbamate derivative was synthesized to overcome this

Ai, Wood, Yang and Welsh
Fig. 6 (a) In vivo efficacy data for niclosamide and its derivative 9 against neuropathic mechanical hyperalgesia through IP route. (b) The integrated effects of the
two compounds were calculated as AUC for 0 to 6 h post-treatment. * P < 0.05 compared with vehicle, data are presented as mean SEM for six animals.
issue. This derivative was inactive against both receptors
in vitro. In order to evaluate the capacity of niclosamide to
reverse neuropathic hyperalgesia, it was tested in a preclinical
model of mechanical NP using PSN ligation on the rear limb
of rats. Three different routes of drug administration (IT, IP,
and PO) were tested to assess in vivo efficacies of niclosamide
and its carbamate derivative. Niclosamide was found to be
substantially active in blocking the mechanical hyperalgesia
associated with nerve injury through all three dosing routes.
The in vivo activity of the carbamate prodrug of niclosamide (9)
was influenced by drug dosing forms which affect its absorp-
tion and, consequently, the systemic concentration of bioac-
tive niclosamide. Compound 9 produced significant anti-
hyperalgesic effects in vivo when administered IP but only min-
imally when administered PO. A plausible explanation for the
ineffectiveness of 9 when administered via PO is poor oral
bioavailability through the GI tract with the current formula-
tion, although this notion would not explain the modest effec-
tiveness of 9 for inflammatory pain (Fig. 1s). The carbamate
pro-moiety of 9 can be released by hydrolytic reaction to yield
bioactive niclosamide in vivo, as demonstrated by effective at-
tenuation of NP in animal models with 9 through IP injection.
Differences in bioavailability between the PO and IP routes
more likely stem from poor oral solubility in the digestive tract,
poor membrane permeability, or a combination of both phe-
nomena that would impair systemic exposure of the drug that
is needed to produce significant in vivo effects. Formulation is
known to be a possible approach to overcome the issue of
bioavailablity for certain drugs. We tested niclosamide carba-
mate derivative with a different vehicle formulation in another
preclinical pain model, the peripheral inflammatory pain
model induced by injection of complete Freund’s adjuvant
(CFA) on the hind paw of rats, in which some NAMs of group
I mGluRs are shown to attenuate the enhanced nociception
and noxious stimulus-induced glutamate release in rats with
inflammation pain in vivo (36). Our preliminary results indicat-
ed that the mechanical hyperalgesia in animals with inflam-
matory pain was significantly ameliorated by the prodrug of
niclosamide (9) with new formulation (Supplemental
Information). Bioanalytical experiments need to be conducted
to compare the pharmacokinetic profiles of niclosamide with
different formulations. The findings from our two pain-related
preclinical models not only provide further evidence for in vivo
efficacy of niclosamide through modulating aberrant gluta-
mate transduction by antagonizing Group I mGluRs, but also
suggest that proper formulation is required to fully realize the
therapeutic potential of this poorly soluble drug.
Since niclosamide is a dual mGluR1/mGluR5 NAM, it
would be interesting to compare its in vivo efficacy with other
selective NAMs, such as MPEP. A substantial body of struc-
tural and neurophysiological evidence and in vivo studies using
animal models suggests that the effects of mGluR1 and
mGluR5 modulation would be similar and perhaps comple-
mentary (11,37). For example, a recent study that compared
the effects of mGluR1-specific NAM (JNJ1656708) and
mGluR5-specific NAMs (MTEP and MPEP) in two

Niclosamide for Neuropathic Pain
Fig. 7 (a) In vivo efficacy data for niclosamide and 9 against neuropathic mechanical hyperalgesia through PO route. (b) The integrated effects of the two
compounds were calculated as AUC for 0 to 6 h post-treatment. (c) The AUC values of niclosamide at five different concentrations. * P < 0.05 compared with
vehicle, data are presented as mean SEM for six animals.
behavioral screening experiments showed very similar ac-
tivity profiles among these agents (38). In addition, the
recent study by Caraci et al. (39) demonstrates dual
mGluR2/3 receptor agonist LY379268 is neuroprotective
against synthetic β-amyloid protein (Aβ) induced toxicity;
however, selective potentiation of the mGluR2 receptor
enhances neuronal vulnerability to Aβ. Thus niclosamide
found in the present study would be a valuable pharma-
cological tool as a dual mGluR1 and mGluR5 NAM with
low nanomolar activity to examine specific or comple-
mentary role of these two receptors in NP and other re-
lated neurological diseases.
CONCLUSION
Taken together, the present study reveals that niclosamide is a
potent Group I mGluR NAM, which may serve as a promising
starting point for subsequent development of effective thera-
peutics for NP. To our best knowledge, this report together
with a concomitant publication by the authors (16) represents
the first instance of niclosamide and its structurally related an-
alogues and derivatives as potential treatments for pain.
ACKNOWLEDGMENTS AND DISCLOSURES
The toxicity profiles of niclosamide are well known,
and the therapeutic dosage of niclosamide is 2000 mg/
day in adults and 1000–1500 mg/day in children when
given orally. However, a complete toxicological profile
of niclosamide and 9 remains to be determined, espe-
cially for those related to neurological and psychiatric
adverse effects, due to the important role of Group I
mGluRs played in glutamatergic transmission.
The authors acknowledge the resources, encouragement and
support provided by Snowdon, Inc. (Monmouth Junction, NJ,
USA). WJW wishes to acknowledge partial support for this
work from the National Institutes of Health-National
Institute for Environmental Health Sciences [P30
ES005022]. NA wishes to acknowledge partial support for this
work from the National Natural Science Foundation of China

Ai, Wood, Yang and Welsh
16. Ai N, Wood RD, Welsh WJ. Identification of Nitazoxanide as a
Group I Metabotropic Glutamate Receptor Negative Modulator
for the Treatment of Neuropathic Pain: An In Silico Drug
Repositioning Study. Pharm Res. 2015;32:2798–807.
17. De Clercq E. Potential antivirals and antiviral strategies against
SARS coronavirus infections. Expert Rev Anti Infect Ther.
2006;4:291–302.
grant [81520988]. The authors declare that they have no
conflict of interests.
Author Contributions Participated in research design: N. Ai,
R.D. Wood, and W.J. Welsh.
Conducted experiments: N. Ai, R.D. Wood, and E. Yang.
Contributed new reagents or analytic tools: R.D. Wood
and W.J. Welsh.
18. Wu CJ, Jan JT, Chen CM, Hsieh HP, Hwang DR, Liu HW, et al.
Inhibition of severe acute respiratory syndrome coronavirus repli-
cation by niclosamide. Antimicrob Agents Chemother. 2004;48:
2693–6.
Performed data analysis: N. Ai, R.D. Wood, E. Yang, and
W.J. Welsh.
Wrote or contributed to the writing of the manuscript: N.
19. Chen W, Chen M, Barak LS. Development of small molecules
targeting the Wnt pathway for the treatment of colon cancer: a
high-throughput screening approach. Am J Physiol Gastrointest
Liver Physiol. 2010;299:G293–300.
Ai and W.J. Welsh.
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